3 revealed that there is no interference

3 Results and discussions

3.1 Optimizing the LC-MS/MS conditions

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To
optimize the LC condition, different mobile phases including MeOH, ACN, ammonia
acetate, and formic acid, and different types of columns including C18,
HSS T3, BEH Amide columns, were tested. Based on the shape of the
peak and the signal response in MS, methanol (containing 0.1% formic acid)/water
(containing 0.1% formic acid) and HSS T3 column were selected as the
mobile and the stationary phases. A gradient
elution was established based on the shape of the MC-A peak to increase the
through-put of the method. In addition, both positive and negative scan mood
were tested. The results showed that positive scan was more
sensitive. Compound and instrument dependent parameters were optimized by
infusing the compound solution into the MS directly using a syringe pump. MRM
scan type was used to improve the specificity. The MS/MS of MC-A and the I.S. are shown in
Fig. 2A and 2B.

3.2. Specificity, linearity, LLOD

The specificity of the method was determined by injecting blank plasma,
blank plasma spiked with MC-A and I.S., and plasma samples from the PK study.
The results revealed that there is no interference at the retention times of the
analyte and I.S. (S/N>3, Fig. 3), indicating the specificity of this method
is acceptable. The standard curves were linear in the
concentration range of 2,000.00-0.49 ng/mL in the plasma. The LLOD was 0.24
ng/mL.

3.5. Recovery and matrix effect

The extraction recoveries were
evaluated using QC samples (n=3) at 0.80, 40.00, 500.00, and 1,500.00 ng/mL. The recovery was > 78.1% (Table 2), suggesting that
this protein precipitation method could
extract MC-A from the plasma. The matrix effect at 0.80, 40.00, 500.00, and
1,500.00 ng/mL were